Erlenmeyer flask or conical flask, usually made of glass, is widely used for laboratory scale suspension culture of microorganism, such as Escherichia Coli and yeast. Due to being non-disposable culture vessel, it has to be cleaned, packaged and sterilized prior to use, which are time, labor and energy consuming work, and has the potential risk of cross-contamination. Despite the rigid plastic conical flasks are possible for single use, they are not economically effective and produce more waste. To overcome these drawbacks, here we successfully developed a novel disposable flexible bioreactor with a plastic film through a thermo-fusion technique. With a triangular pyramid shape, the bioreactor enabled itself to keep a three-dimensional internal space without needing air inflation and well adapted to the commercial available orbital shaker. Unlike the conventional rigid conical flasks and other reported flexible flasks, which had to be fixed in the shaker, the flexible bioreactor could keep sitting on the silicone pad-carpeted platform of the orbital shaker steadily without any fixation needed at the shaking speeds below 250 rpm, thus making it simple to handle. Compared with the traditional conical glass flasks, the innovative flexible bioreactors achieved a significant higher efficiency in bacteria growth and oxygen transfer rates. In conclusion, the novel flexible bioreactor is an ideal disposable culture vessel for microorganism suspension culture at laboratory scale and holds a promising potential to replace the glass flask and rigid plastic flask in the future.